Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast.
In order to facilitate efficient purification of the plant plasma membrane H+-ATPase expressed in yeast, a recombinant H+-ATPase protein with an N-terminal affinity tag of six histidine residues was engineered. When expressed in yeast the recombinant protein accumulated in the endoplasmic reticulum in an active form and showed characteristics comparable with those of the wildtype plasma membrane H+ATPase (Km,ATP, 1.1 mM; pH optimum, 6.6). After solubilization of the membrane proteins from the endoplasmic reticulum with n-dodecyl-beta-d-maltoside, the recombinant protein was purified under nondenaturing conditions by chromatography on Ni2+-nitriloacetic acid-agarose. A fraction was obtained which contained 4.2% of the initial amount of the protein and 26.6% of the ATPase-activity present in the endoplasmic reticulum. The purified protein has a specific activity of 32.6 micromol min-1 mg protein-1 at pH 6.5 and 30 degrees C. This rate is equivalent to a molecular activity of 3400 min-1. The purified plasma membrane H+-ATPase could be reconstituted into liposomes and demonstrated in this configuration the ability to pump protons. The method proves to be a convenient and rapid method for the preparation of purified single isoforms and mutant proteins of the plant plasma membrane H+-ATPase in high and functional quantities. This method might also be useful for achieving purification of other P-type ATPases, normally expressed at very low levels in heterologous systems.
Lanfermeijer
F C
FC
Institute of Molecular Biology, Copenhagen University, Oster Farimagsgade 2A, Copenhagen K, DK-1353, Denmark.
Venema
K
K
Palmgren
M G
MG
D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D010944:Plants / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000596:Amino Acids; D008081:Liposomes; D011994:Recombinant Proteins; D006639:Histidine; D006180:Proton-Translocating ATPases